QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) was used to extract genomic DNA according to the manufacturer’s instructions. The extracted DNA was stored at −30 °C until used in the PCR amplification. The presence of E. bieneusi DNA was confirmed by amplifying of an approximately 390 bp fragment including the internal transcribed spacer (ITS) region of the rRNA gene. The primers and PCR thermal cycler parameters used in the present study have been described in our previous study [5 (link)]. Briefly, PCR mixtures (25 μL) were composed of 12.5 μL of Taq mix (Promega, Madison, WI, USA), 1 μL each of the forward and reverse primers (10 μM) (Sunny Biotechnology, Shanghai, China), 1 μL of DNA template and 9.5 μL of nuclease-free water (Promega, Madison, WI, USA). The positive control (E. bieneusi-positive DNA) and negative control (nuclease-free water) samples were added in each PCR run. All the samples were conducted by PCR for three times to make sure the authenticity of the results. The PCR products were examined by 2% agarose gel electrophoresis with GelRed staining (Biotium Inc., Hayward, CA, USA) and observed using a gel imaging system.
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